SCCS/1358/10

Scientific Committee on Consumer Safety
SCCS

Basic criteria for the in vitro assessment of dermal
absorption of cosmetic ingredients

The SCCS adopted this opinion at its 7th plenary meetingof 22 June 2010

SCCS/1358/10
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________

About the Scientific Committees
Three independent non-food Scientific Committees provide the Commission with thescientific advice it needs when preparing policy and proposals relating to consumer safety,
public health and the environment. The Committees also draw the Commission's attention
to the new or emerging problems which may pose an actual or potential threat.
They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee
on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and
Newly Identified Health Risks (SCENIHR) and are made up of external experts.
In addition, the Commission relies upon the work of the European Food Safety Authority(EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre for
Disease prevention and Control (ECDC) and the European Chemicals Agency (ECHA).
SCCSThe Committee shall provide opinions on questions concerning all types of health and safety
risks (notably chemical, biological, mechanical and other physical risks) of non-foodconsumer products (for example: cosmetic products and their ingredients, toys, textiles,
clothing, personal care and household products such as detergents, etc.) and services (forexample: tattooing, artificial sun tanning, etc.).
Scientific Committee members
Jürgen Angerer, Ulrike Bernauer, Claire Chambers, Qasim Chaudhry, Gisela Degen, Gerhard
Eisenbrand, Thomas Platzek, Suresh Chandra Rastogi, Vera Rogiers, Christophe Rousselle,
Tore Sanner, Kai Savolainen, Jacqueline Van Engelen, Maria Pilar Vinardell, Rosemary
Waring, Ian R. White
Contact
European Commission
Health & Consumers
Directorate C: Public Health and Risk Assessment
Unit C7 - Risk Assessment
Office: B232
B-1049 Brussels
Sanco-Sc6-Secretariat@ec.europa.eu© European Union, 2010
ISSN 1831-4767
doi:10.2772/25843

ISBN 978-92-79-12747-2
ND-AQ-09-019-EN-N

The opinions of the Scientific Committees present the views of the independent scientists
who are members of the committees. They do not necessarily reflect the views of theEuropean Commission. The opinions are published by the European Commission in their
original language only.
http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

2SCCS/1358/10
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________ACKNOWLEDGMENTS

Prof. G. Degen
Prof. T. Platzek
Dr. S.C. Rastogi
Prof. V. Rogiers
Prof. T. SannerDr. J. van Engelen
Prof. M.P. Vinardell(rapporteur)(chairman)

Keywords: SCCS, scientific opinion, basic criteria, in vitro, dermal absorption, cosmetic
ingredients, directive 76/768/ECC

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), basic criteria for
the in vitro assessment of dermal absorption of cosmetic ingredients, 22 June 2010

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SCCS/1358/10
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________TABLE OF CONTENTSACKNOWLEDGMENTS ........................................................................................... 3
1.

BACKGROUND ............................................................................................. 52.

GENERAL PRINCIPLES................................................................................... 6

3.

PRINCIPLE OF THE TEST ............................................................................... 7

4.

METHODOLOGY AND FACTORS AFFECTING DERMAL ABSOROPTION .................... 7

5.

CONCLUSION ............................................................................................ 12

6.

REFERENCES ............................................................................................. 12

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________

1. BACKGROUND
As early as June 1999, before any OECD guideline on percutaneous absorption wasavailable, important scientific articles on this methodology were published [Beck et al. 1995,
Diembeck et al. 1999]. The SCCNFP discussed the scientific progress with external experts
in the field and subsequently adopted its first "Basic criteria for the in vitro assessment of
percutaneous absorption of cosmetic ingredients" [SCCNFP/0167/99]. The publication ofsuch a set of criteria was seen as a pro-active action of the SCCNFP in order to support andspeed up the introduction of in vitro dermal absorption studies of cosmetic ingredients inthe evaluation of dossiers of cosmetic ingredients belonging to Annexes III, IV, VI or VII to
Dir. 76/768/EEC. Moreover, the opinion did not only provide basic criteria for the in vitro
assessment of dermal absorption of cosmetic ingredients, but also discussed some generalprinciples and practical aspects of the methodology.
In December 2000, two draft OECD Guidelines on dermal absorption became available,
namely 1) OECD Draft Guideline 427 on skin absorption: in vivo method [OECD 427], and
2) OECD Draft Guideline 428 on skin absorption: in vitro method [OECD 428]. The
combination of the OECD-publication with gained practical experience, has led to theincorporation of the in vitro dermal absorption study in toxicological dossiers of cosmetic
ingredients,
plant
protection
products,
biocides,etc[98/8/EC,
2000/6/EC,
Sanco/222/2000].
Subsequently, in order to provide an up to date guidance for the application of the in vitro
methodology for cosmetic ingredients, document SCCNFP/0167/99 was revised and updated
in 2003 [SCCNFP/0750/03]. Existing documents such as the "Proposed rule for in vitro
dermal absorption rate testing …" [US EPA 1999], the "Guidance document on dermal
absorption" [Sanco/222/2000], the "Draft Guidance Document for the conduct of skinabsorption studies" [OECD 2000], and the "Technical Guidance Document on Risk
Assessment part 1" [ECB 2003] were taken into consideration.
The SCCP noticed later that several dossiers failed to fulfil the requirements as described in
document SCCNFP/0750/03. In particular, a revision of all hair dye dossiers revealed anumber of problems [Pauwels and Rogiers 2004]. An expert group was then established by
SCCP with the purpose to update the "SCCP basic criteria for the in vitro assessment ofdermal absorption of cosmetic ingredients" and to resolve the identified problems. On thebasis of the recommendations of this expert group, the document “SCCP basic criteria for
the in vitro assessment of dermal absorption of cosmetic ingredients" was updated in 2006
[SCCP/0970/06] and the chapter on dermal absorption in the SCCP Notes of Guidance, 6th
revision was expanded.
Meanwhile, OECD Guideline 428 was finalized in 2004 and four years later, the protocol was
copied into EC method B.45 [EU, 2008].
The application of the 2006 update of the “SCCP basic criteria for the in vitro assessment ofdermal absorption of cosmetic ingredients" for the evaluation of the dermal absorption
studies in dossiers submitted by the Industry, frequently resulted in disagreement on thenumber of skin samples and donors, concentrations to be tested, variability of the test
results and the value to be used for the calculation of Margin of Safety (MoS). This led to
non-acceptance of several submitted studies [Rogiers and Pauwels 2008].
Above-mentioned disagreements and the use of default values <100% for dermal
absorption in the absence of appropriate experimental data, were discussed among experts
from SCCP, COLIPA, as well as external experts in a ”Special meeting on dermal absorption
issues” in October 2008. The conclusions of this meeting are incorporated in the present
updated version of the “SCCP basic criteria for the in vitro assessment of dermal absorptionof cosmetic ingredients" and will be included in the next revision of the SCCS Notes of
Guidance.
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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________The dermal absorption of nanoparticles is not covered by this opinion.

2. GENERAL PRINCIPLESDefinitions
The definitions with respect to dermal absorption slightly diverge in different official
documents. Hereunder, the definitions as proposed by the WHO [WHO 2005] are given:
The percutaneous/dermal absorption process is a global term which describes thepassage of compounds across the skin. This process can be divided into three steps:-

penetration, which is the entry of a substance into a particular layer or structuresuch as the entrance of a compound into the stratum corneum;-

permeation, which is the penetration through one layer into another, which is both
functionally and structurally different from the first layer;-

resorption which is the uptake of a substance into the vascular system (lymph and/or
blood vessel), which acts as the central compartment.

The purpose of dermal absorption studies of cosmetic ingredients is to obtain qualitative
and/or quantitative information on the amounts that may enter, under in-use conditions,
into the systemic compartment of the human body. These quantities can then be taken into
consideration to calculate the margin of safety using the NOAEL of an appropriate repeateddose toxicity study with the respective substance.
Justification for the use of in vitro dermal absorption studies on isolated skin is based on thefact that the epidermis, in particular the stratum corneum, forms the principal in vivo
barrier of the skin against penetration and uptake of xenobiotics in the body.
Under in vivo conditions, the microcirculatory system (blood and lymph vessels) may carrycompounds from the dermis into the central compartment (resorption). In vitro, themicrocirculation is compromised, thus the potential resorption of a compound cannot beadequately studied in such a setting.
The dermal tissue may retain penetrating compounds that, in vivo, would have been
removed into the systemic compartment. Thus, either the dermis must be removed prior toin vitro investigations (so-called split-thickness skin) or possible in vitro retention in thedermis and living epidermis (without stratum corneum) must be taken into account wheninterpreting the in vitro results.
The epidermis renews by continuous outward proliferation, differentiation anddesquamation. About one layer of corneocytes is shed off per day. After topical application,
xenobiotics detected in vitro in the skin, particularly in the stratum corneum and thepilosebaceous units, might in vivo have been lost from the skin via desquamation or sebum
secretion, respectively. Because these processes are not present in vitro, the final epidermal(stratum corneum) levels in vitro could be elevated compared with the corresponding invivo levels. Therefore, although the EU B.45 method defines the ‘absorbable dose’ as theamount present on or in the skin following washing, the amount of substance present in thestratum corneum is not considered systemically available.
According to the above principles, the following should be included in the protocol for invitro dermal absorption studies:

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________i.

Studies should be performed on appropriate standardised skin preparations. The
respective choice should be justified in the protocol. The WHO recommends humanskin as the gold standard.

ii.

At the end of the experiment, a full mass balance should be performed.

iii.

When considerable cutaneous metabolism of the test compound occurs in vivo, further
studies may be necessary. It should be noticed that frozen skin preparations may lackthe enzyme systems for biotransformation of the test compound and may not provide
an accurate picture of the formation of metabolites and their dermal absorption.
Therefore in vitro studies using frozen skin may not provide complete information on
the dermal absorption of compounds that undergo biotransformation in the skin nor on
their potential metabolite(s) formed. The role of cutaneous biotransformation in theabsorption process is still a matter of scientific debate.

iv.

Sometimes an irreversible binding of an ingredient to the epidermis may occur,
followed by elimination through in vivo desquamation of the skin surface. When thismechanism is assumed, it must be documented by separate experiments.

3. PRINCIPLE OF THE TESTAt present only skin preparations of natural origin may be used. Although the quality ofcultured or reconstituted skin has improved importantly during the last years [Bouwstra etal. 2008], these cultures do not possess a complete barrier function comparable to that ofliving skin.
OECD Guideline 428 / EC B.45 should be followed as close as possible, taking into accountthe guidance given here. Any deviation from this guideline should be documented andjustified by appropriate scientific argumentation.
The test substance should be applied in an appropriate formulation on the skin sample
which is placed in a diffusion cell (cf. 4.1). The skin is then positioned between the upperand lower chambers of the cell.
Diffusion cells may be of static or flow-through design. The integrity of the barrier should bechecked by an appropriate method. The test sample should remain in contact with the skinon the donor side for a defined period of time, corresponding to the typical use of thecosmetic end product, such as leave-on or rinse-off conditions. The receptor fluid should besampled at an early time point (e.g. after 30 minutes), at the end of the experiment and at
appropriate time points in between in order to obtain an absorption-time profile. The skinand/or fluid samples should be analysed by appropriate and validated analytical methods,
such as liquid scintillation counting, HPLC, GC or other suitable methods. Information on thesensitivity and repeatability / time-different intermediate precision of the analyticalmethod(s) should be provided.

4. METHODOLOGY AND FACTORS AFFECTING DERMAL ABSOROPTION
Dermal absorption can be affected by several factors: e.g. physical and chemical propertiesof the substance, type and composition of the formulation, occlusion, concentration of thesubstance in the formulation, exposure pattern, skin site of the body and technical aspectsof the respective in vitro test [Howes et al. 1996, Schaefer and Redelmeier 1996, ECETOC
1993].

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SCCS/1358/10
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________In the following section, an overview is given of factors that may affect dermal absorption inin vitro dermal absorption studies.
4.1. Diffusion cell design
The diffusion cell consists of an upper donor and a lower receptor chamber, separated by
the skin preparation under investigation. The stratum corneum faces the donor chamber.
Diffusion cells should consist of inert non-adsorbing material. Temperature control of thereceptor fluid is crucial throughout the experiment. The skin surface temperature in the
diffusion cell should be kept at the in vivo skin temperature of 32 ± 1°C. Additional dermal
absorption studies may be required in some specific cases, e.g. when substance exposure ata higher skin temperature may be expected. The advantage of using a flow-through system
is continuous sampling; the advantage of using a static system is the increase in sensitivity
for test substances only poorly penetrating the skin.
4.2. Receptor fluid
The composition of the receptor fluid is chosen so that it does not limit the extent ofdiffusion of the test substance, i.e. the solubility and the stability in the receptor fluid of thechemical under investigation have to be guaranteed. Saline or buffered saline solutions arecommonly used for hydrophilic compounds. For lipophilic molecules, serum albumin orappropriate solubilisers / emulsifiers are added in amounts which must not interfere withmembrane integrity. The fluid should not interfere with the analytical procedure.
As a general rule, the receptor fluid should have a physiological pH. Any deviation from thisprinciple should be justified. E.g. in the case of 50/50 ethanol/water, as proposed in theOECD Guideline [OECD 2004], evidence should be included in the dossier, showing that thisdoes not significantly affect the integrity of the skin.
In order to avoid serial non-detects at early sample points, the receptor fluid volume should
be kept to a minimum.
The receptor fluid, preferably degassed in order to avoid formation of air bubbles during theexperiment, should be thoroughly stirred (static cells) or continuously replaced (flowthrough cells) during the entire experiment. The choice of static or flow-through conditionsin the receptor cell should be made on a compound-by-compound basis, depending on its
theoretical absorption properties and the objective of the study. The choice of the test
system should be justified in the study report. The amount of penetrated substance in thereceptor fluid should not exceed 10% of its saturation level at any time, in order tominimise interference with the free diffusion process that could produce an underestimationof dermal absorption. The substance should be stable in the receptor fluid for the durationof the in vitro test and the subsequent analysis.
4.3. Skin preparations
Human skin is the best choice but is not always readily available. Alternatively, pig skin may
be used because it shares essential permeation characteristics with human skin. Rat skin is
2 to 10 times more permeable than human skin [Ross et al. 2000] and it is therefore notrecommended.
The use of cultured or reconstructed human skin models is under development and thosesystems are not yet advised for in vitro testing on the basis of their insufficient barrier
function [Bouwstra et al. 2008].
The following information is required:
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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________-

Origin of skin samples used.
Species: by preference human or pig skin should be used.
Skin location: abdomen, leg or breast (human skin); abdomen, breast, back, flanks orears (pig skin).
Gender and age: although these factors are not believed to be important variables,
they should be stated.
Fresh/frozen skin: when significant biotransformation of the test compound in the skinis expected, freshly excised, viable skin should be used [Diembeck et al. 1999].
Details on preservation and storage conditions of the skin should be specified (e.g.
skin can be stored in aluminium foil at -20°C or lower) [Howes et al. 1996, Bronaugh
et al. 1986]. During transport skin samples should be kept at or below 4°C.
Numbers of skin samples and donors (see also point 4.13.)

Skin samples that may be used are split-thickness (200-500 µm) or full-thickness(500-1000 µm) skin preparations [Sanco/222/2000]. Dermatomed skin is often used. Skin
thickness should be measured by an appropriate method, which should be described in thereport. The skin samples should be prepared to fit the experimental cell.
-

For human skin:-

For pig skin:split-thickness skin should be the general rule. If for a specificreason, full-thickness is required, this should be justified.
since it is technically more difficult to obtain intact split-thickness
skin, this could justify the use of full-thickness skin.

When epidermal membranes are used for the in vitro dermal absorption study, the reason
for this should be justified. Epidermal membranes are sometimes quite fragile and somemass balance techniques (e.g. tape stripping) cannot be applied to this model. It must also
be mentioned that epidermal membranes may overestimate human in vivo skin absorption[Van de Sandt et al. 2000].
The minimum skin area to be covered is considered to be 0.64 cm².
4.4. Skin integrity
Barrier integrity is crucial for the experiment, and must therefore be measured. This isachieved by either measuring the penetration of a marker molecule, e.g. tritiated water,
caffeine or sucrose, or by physical methods, such as determination of TEWL
(Transepidermal Water Loss) or TER (Transcutaneous Electrical Resistance). Data obtained
should be reported.
4.5. Skin temperature
Because the rate and extent of skin absorption is temperature-dependent, the skin disctemperature should be maintained constant (32 ± 1°C, corresponding to the normal humanskin surface temperature). The method of temperature maintenance should be described in
the report.
4.6. Test substance
The relevant physical and chemical data (e.g. MW, log Pow, solubility, stability, and pKa of
the test substance) should be given.
The purity of the test substance should be described and should be comparable to that of
the substance in marketed products (see 4.10). It is recognised that synthesis of radio9

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________

labelled (i.e. 14C- or 3H-labelled) substances may result in a slightly different purity and/or
impurity profile than that of substances produced by large-scale chemical production. These
differences should be reported.
As mentioned under 4.2, the solubility and stability of the test substance in the receptor
fluid for the entire test duration should be documented.
4.7. Preparation of the dose and vehicle / formulation
The dose and vehicle / formulation should be representative for the in use condition(s) of
the finished cosmetic product. The quantitative composition of every formulation usedduring the experiment should be given.
For oxidative hair dyes, the relevant combination(s) of hair dye precursor(s), coupler(s) anddeveloper(s) should be tested.
Several concentrations, including the highest concentration of the test substance in a typicalformulation should be studied.. These concentrations should be selected in such a way thatthe range of the linear curve of concentration versus dermal absorption is demonstrated.
The stability of the test substance under the foreseeable conditions of application and usage
must be ascertained.
4.8. Dose and volume of test substanceThe dose of the test formulation as well as its contact time (exposure) with the skin should
resemble use conditions. The amount of the formulation to be applied should be adapted tothe consumer use/technical conditions. The values to be used are described in the Notes of
Guidance: 2-5 mg/cm2 for solids and semi-solid preparations, and up to 10 µl/cm2 for
liquids. For oxidative hair dye formulations, 20 mg/cm2 is applied. Deviations should be
explained. The volume of formulation used should be appropriate to spread the sample
homogeneously over the skin surface. This depends on the viscosity and lipophilicity of theformulation. Both mass and volume applied should be stated in the test report.
4.9. Study period and sampling
The exposure time and sampling period(s) should be defined in the protocol. The normal
exposure time is 24 hours with regular sampling intervals. Longer duration of the study may
result in membrane deterioration and requires careful control of membrane integrity. The
exposure time should be consistent with the intended use of the cosmetic formulation. E.g.
for oxidative hair dye formulations, the time of contact could vary according to the inmarket use. The SCCS recommends at least 30 minutes contact in dermal absorption
testing. The skin surface will then be rinsed using a procedure mimicking the consumer
situation. Sampling of the receptor fluid is continued until e.g. 24 hours.
The frequency of sampling should be chosen adequately to allow the determination of theextent/rate of absorption and the absorption profile. In order to estimate absorption
kinetics, samples should be obtained from at least 6 post-application time points, including
one early time point (30 minutes). For rinse-off products, measurements after rinsing haveto be taken. The full sampling procedure must be described in the report.
4.10.

Analytical methods

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________

Appropriate analytical techniques, e.g. liquid scintillation counting, HPLC or GC, should be
used. Their validity, sensitivity and detection limits should be documented in the report.
When high sensitivity is required, the test substance should, whenever possible, be radiolabelled.
Qualitative or semi-quantitative methods, such as micro-autoradiography, may be usefultools for skin distribution assessments.
4.11.

Data collection

The test compound must be determined in the following compartments:-

Product excess on the skin (dislodgeable dose)
Stratum corneum (e.g. adhesive tape strips)
Living epidermis (without stratum corneum)
DermisReceptor fluid

To calculate the mass balance correctly, it is also necessary to measure the amounts of test
substance adsorbed to the equipment (included in rinsing solutions and/or compartments).
4.12.

Mass balance analysis / recovery

The mass balance of the applied dose must be determined.
The overall recovery of test substance (including metabolites) should be within the range of
85-115%. Lower or higher recovery rates should be investigated and/or explained.
4.13.

Variability / validity / reproducibility

The technical ability of the performing laboratory and the validity of the method used should
be assessed at regular intervals, at least twice per year, by using reference compounds like
caffeine or benzoic acid. These data should be included in the study report [OECD 2004,
Van de Sandt et al. 2004].
Factors that affect the variability in the dermal absorption of a test substance are:- Inter-individual and intra-individual characteristics of the stratum corneum barrier,- The variation in various parameters, such as skin temperature, skin thickness,
vehicle, concentration of applied substance, amount of applied formulation, andexposure duration- The use of static or flow through cell system- The uncertainty of the measurement of the test substance.
During a meeting with industry in October 2008, the variability issue was discussed indetail, especially in view of the outcome to be used for further calculations and in view of
the number of required skin samples. Based on this discussion, the following decision wasmade by the SCCS: for a reliable dermal absorption study, 8 skin samples from at least 4
donors should be used.
When studies correspond to all of the basic requirements of the SCCS, the mean + 1SD will
be used for the calculation of the MoS. The reason for not using the mean per se is thefrequently observed high variability in the in vitro dermal absorption assays. Moreover, themethod was validated based upon practical experience only and did not go through theelaborated validation process as we know it today.
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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredient___________________________________________________________________________________________

In case of significant deviations from the protocol and/or very high variability, the mean +2SD will be used as dermal absorption for the calculation of the margin of safety.

5. CONCLUSION
Dermal absorption can be expressed as an absolute amount [µg/cm2 of skin surface] and/or
as a percentage of the amount of test substance contained in the intended dose applied per
square centimetre of skin surface.
In a classical in vitro dermal absorption setting, the amounts of penetrated substance(s)found in the receptor fluid are considered to be systemically available. Both the epidermis(except for the stratum corneum) and dermis are considered as a sink, wherefore theamounts found in these tissues are considered as absorbed and are added to those found in
the receptor fluid. The amounts that are retained by the stratum corneum at the time ofsampling are not considered to be dermally absorbed, and thus they are not expected tocontribute to the systemic dose.
The absorption rate and mass balance should be calculated separately for each diffusion
cell. Considering skin samples as the main contributor of the variability in results of adermal absorption study, the mean and SD of the dermal absorption rate should be
calculated from at least 8 evaluable results representative of skin from at least 4 donors. All
measurements, statistical processing and obtained kinetic curves should be provided.
The SCCS considers the following criteria as critical for the decision if a study fulfils therequirements:- the availability of 8 evaluable samples originating from 4 donors (for studies
performed previous to this decision, 3 donors may be accepted);- a mass balance of the applied dose which shows to be ≥ 85%;
- a clear mention of the relative standard deviation (RSD or CV) of the measured dermal
absorption rate;- thorough consideration of factors 4.1 to 4.11 as described in the current opinion.
When studies correspond to all of the basic requirements of the SCCS, the mean + 1SD will
be used for the calculation of the MoS. The reason for not using the mean per se is thefrequently observed high variability in the in vitro dermal absorption assays. Moreover, themethod was validated based upon practical experience only and did not go through theelaborated validation process as we know it today.
In case of significant deviations from the protocol and/or very high variability, the mean +2SD will be used as dermal absorption for the calculation of the margin of safety.
In case the results are derived from an inadequate in vitro study, 100% dermal absorption
is used. However, in case MW > 500 Da and log Pow is smaller than -1 or higher than 4, thevalue of 10% dermal absorption is considered [ECHA 2008].

6. REFERENCES
2000/6/EC - Twenty-fourth Commission Directive 2000/6/EC of 29 February 2000 adapting
to technical progress Annexes II, III, VI and VII to Council Directive 76/768/EEC on theapproximation of the laws of the Member States relating to cosmetic products. Official
Journal L 56, 01/03/2000 p.42.
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Beck H., Benech-Kieffer F., Bracher M., Diembeck W., Dupuis J., Lovell W., Steiling W.
Cosmetic ingredients : Guidelines for percutaneous absorption / penetration.COLIPA
Guidelines, 1-36 (1995).
Bouwstra J.A., Groenink H.W., Kempenaar J.A., Romeijn S.G., Ponec M. Water distribution
and natural moisturizer factor content in human skin equivalents are regulated byenvironmental relative humidity. Journal of Investigative Dermatology 128(2):378-88(2008).
Bronaugh R.L., Stewart R., Simon M. Methods for in vitro percutaneous absorption studies
VII : use of excised human skin. Journal of Pharmaceutical Sciences 75, 1094-1097(1986).
Diembeck W., Beck H., Benech-Kieffer F., Courtellemont P., Dupuis J., Lovell W., Paye M.,
Spengler J. and Steiling W. Test guidelines for in vitro assessment of dermal absorption
and percutaneous penetration of cosmetic ingredients. Food & Chemical Tox., 37, 191205 (1999).
Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998
concerning the placing of biocidal products on the market Official Journal L 123, 24/4/98
p. 1.
ECB (European Chemicals Bureau) Technical Guidance Document on Risk Assessment insupport of Commission Directive 93/67/EEC on Risk Assessment for new notified
substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for existing
substances and Directive 98/8/EC of the European Parliament and of the Council
concerning the placing of biocidal products on the market. Doc. EUR 20418 EN/1,
European Communities (2003).
ECHA 2008. Guidance on information requirements and chemical safety assessment.
Chapter R.7c: Endpoint specific guidance. Available through:http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_r7
c_en.pdf?vers=20_08_08 (consulted June 2010).
EU B.45 - Skin absorption - In vitro method. Council Regulation (EC) No 440/2008 of 30
May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of theEuropean Parliament and of the Council on the Registration, Evaluation, Authorisation
and Restriction of Chemicals (REACH). OJ L142, 31 May 2008, 438-443 (2008).
ECETOC Percutaneous absorption. European Centre for Ecotoxicology and Toxicology ofChemicals (ECETOC), Monograph No 20, Brussels (1993).
Howes D., Guy R., Hadgraft J., Heylings J., Hoeck U., Kemper F., Maibach H., Marty J-P.,
Merk H., Parra J., Rekkas D., Rondelli I., Schaefer H., Täuber U. and Verbiese N.
Methods for assessing percutaneous absorption, ECVAM Workshop Report n. 13.
Alternatives To Laboratory Animals 24, 81-106 (1996).
OECD 427 - OECD Guideline for testing of chemicals - Guideline 427 : Skin absorption: In
vivo method Organization for Economic Cooperation and Development, Paris, adopted
13 April 2004.
OECD 428 - OECD Guideline for testing of chemicals - Guideline 428 : Skin absorption: In
vitro method Organization for Economic Cooperation and Development, Paris, adopted
13 April 2004.
OECD Guidance Document for the Conduct of Skin Absorption Studies. Organization for
Economic Cooperation and Development (OECD), Environment Directorate, OECD
Environmental Health and Safety Publications, Series on Testing and Assessment No. 28,
Paris (2004).
Pauwels M. and Rogiers V. How to improve the quality of a European cosmetic ingredients'
dossier? Oral presentation given during the Scientific Forum of the COLIPA (TheEuropean Cosmetic Toiletry and Perfume Association) General Assembly 2004,
Amsterdam, 24/06/2004.
Rogiers V., Pauwels M. (Eds.), 2008. Current Problems in Dermatology 36: Safety
Assessment of Cosmetics in Europe. S. Karger, Basel, Switzerland. ISBN 978-3-80558655-9.
Ross J.H., Dong M.H. and Krieger M.I. Conservatism in pesticide exposure assessment.
Regulatory Toxicology and Pharmacology 31, 53-58 (2000).

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Sanco/222/2000 – Guidance Document on Dermal Absorption European Commission, Healthand Consumer Protection Directorate-General Doc. Sanco/222/2000 rev 6, of
27 November 2002.
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